DNA damage and repair are fundamental to human health and disease. The long-term objectives of this application are to: expand our understanding of the role of DNA helical distortions in DNA damage recognition and processing; determine the factors influencing DNA structure-induced genetic instability; elucidate potential mechanisms involved in translocations associated with certain cancers; and further the development of novel approaches to reduce genetic instability in human cells. In the short term, we will pursue our recent discovery that helical distortions induced by naturally occurring Z-DNA and H-DNA structures are highly mutagenic and can induce DNA double-strand breaks (DSBs) in mammalian cells. We propose to study the effect of DNA helical distortions on genomic instability in plasmid-based systems as well as on chromosomes in human cells and in transgenic mutation-reporter mice. We will focus on the H- DNA-forming sequence located near the translocation breakpoint in the human c-MYC promoter and the Z- DNA sequence located at a chromosomal breakpoint in the human BCL-2 gene, found in lymphomas and leukemias. We will determine the role(s) of DNA repair, replication and transcription in the structure-induced genetic instability. We will use our expertise in the introduction of site-specific DNA helical distortions in the form of well-defined intermolecular triplex structures to test our hypothesis that certain types of DNA helical distortions (in the presence or absence of DNA damage per se) are recognized by the DNA repair machinery in human cells. The new information obtained from these studies will provide insight into the mechanisms of non-B DNA-induced genetic instability; the rate-limiting step in human DNA repair (i.e.distortion/damage recognition); and the overlap between nucleotide excision repair and mismatch repair in processing DNA helical distortions. It will also identify the proteins involved in the generation of DSBs induced bynon- canonical DNA structures formed at sequences that map to translocation breakpoints in human cancers. These discoveries should lead to a better understanding of the pathogenesis of cancers and other diseases that are caused by DNA damage and naturally occurring helical distortions, and ultimately to the development of new approaches to treatment and prevention.